[Off-line comprehensive two-dimensional countercurrent chromatography-liquid chromatography separation of Curcuma...

The chemical constituents of volatile oils used in traditional Chinese medicine are highly complex. Thus, achieving the complete separation of volatile oils by one-dimensional chromatography is difficult owing to the low peak capacity of the technique. Although comprehensive two-dimensional gas chromatography provides an efficient means for separating volatile oils, it cannot be used to screen bioactive components because of its limitations. Therefore, developing a new method to separate volatile oils based on liquid chromatography is of great significance in efforts to obtain new approaches to screen bioactive components in volatile oil. The objectives of the present study are to establish an efficient method for separating the chemical constituents of Curcuma volatile oil using off-line comprehensive two-dimensional countercurrent chromatography-liquid chromatography (CCC-LC) and to investigate the two-dimensional peak capacity, orthogonality, and spatial coverage of this method. Both CCC and LC conditions were optimized. A biphasic n-hexane-methanol-water solvent system was selected via the colorimetric method, and the lower phase was used as the mobile phase in gradient-elution mode: 0-55 min, n-hexane-methanol-water (5∶2∶3 v/v/v); 55-170 min, n-hexane-methanol-water (5∶3∶2, v/v/v); 170-290 min, n-hexane-methanol-water (5∶4∶1, v/v/v). After gradient elution, elution-extrusion elution mode was applied within 290-375 min. Good resolution was achieved by the CCC separation process. The HPLC separation process was carried out with gradient elution using a mobile phase composed of acetonitrile (A)-water (B): 0-10 min, 50%A-65%A; 10-14 min, 65%A; 14-21 min, 65%A-85%A; 21-25 min, 85%A-95%A; 25-30 min, 95%A-55%A; 30-40 min, 55%A. Curcuma volatile oil was separated under the above optimized two-dimensional separation conditions, and the data obtained were drawn into a two-dimensional contour map using Matlab software. The calculated total peak capacity exceeded 954, which was 10 times more than that of one-dimensional chromatography. High orthogonality (r=0.17) and spatial coverage factor (68.1%) were also obtained. Our research provides a new methodology for separating volatile oils used in traditional Chinese medicine as well as an approach for evaluating the quality of traditional Chinese medicinal herbs using two-dimensional chromatographic fingerprints.